affinity chromatography

chromatography

[kro″mah-tog´rah-fe]

a technique for analysis of chemical substances. The term chromatography literally means color writing, and denotes a method by which the substance to be analyzed is poured into a vertical glass tube containing an adsorbent, the various components of the substance moving through the adsorbent at different rates of speed, according to their degree of attraction to it, and producing bands of color at different levels of the adsorption column. The term has been extended to include other methods utilizing the same principle, although no colors are produced in the column. adj., adj chromatograph´ic.

The mobile phase of chromatography refers to the fluid that carries the mixture of substances in the sample through the adsorptive material. The stationary or adsorbent phase refers to the solid material that takes up the particles of the substance passing through it. Kaolin, alumina, silica, and activated charcoal have been used as adsorbing substances or stationary phases.

Classification of chromatographic techniques tends to be confusing because it may be based on the type of stationary phase, the nature of the adsorptive force, the nature of the mobile phase, or the method by which the mobile phase is introduced.

The technique is a valuable tool for the research biochemist and is readily adaptable to investigations conducted in the clinical laboratory. For example, chromatography is used to detect and identify in body fluids certain sugars and amino acids associated with inborn errors of metabolism.

adsorption chromatography that in which the stationary phase is an adsorbent.

affinity chromatography that based on a highly specific biologic interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand. Any of these substances, covalently linked to an insoluble support or immobilized in a gel, may serve as the sorbent allowing the interacting substance to be isolated from relatively impure samples; often a 1000-fold purification can be achieved in one step.

column chromatography the technique in which the various solutes of a solution are allowed to travel down a column, the individual components being adsorbed by the stationary phase. The most strongly adsorbed component will remain near the top of the column; the other components will pass to positions farther and farther down the column according to their affinity for the adsorbent. If the individual components are naturally colored, they will form a series of colored bands or zones.

Column chromatography has been employed to separate vitamins, steroids, hormones, and alkaloids and to determine the amounts of these substances in samples of body fluids.

exclusion chromatography that in which the stationary phase is a gel having a closely controlled pore size. Molecules are separated based on molecular size and shape, smaller molecules being temporarily retained in the pores.

gas chromatography a type of automated chromatography in which the mobile phase is an inert gas. Volatile components of the sample are separated in the column and measured by a detector. The method has been applied in the clinical laboratory to separate and quantify steroids, barbiturates, and lipids.

gas-liquid chromatography gas chromatography in which the substances to be separated are moved by an inert gas along a tube filled with a finely divided inert solid coated with a nonvolatile oil; each component migrates at a rate determined by its solubility in oil and its vapor pressure.

ion exchange chromatography that utilizing ion exchange resins, to which are coupled either cations or anions that will exchange with other cations or anions in the material passed through their meshwork.

paper chromatography a form of chromatography in which a sheet of blotting paper, usually filter paper, is substituted for the adsorption column. After separation of the components as a consequence of their differential migratory velocities, they are stained to make the chromatogram visible. In the clinical laboratory, paper chromatography is employed to detect and identify sugars and amino acids.

partition chromatography a process of separation of solutes utilizing the partition of the solutes between two liquid phases, namely the original solvent and the film of solvent on the adsorption column.

thin-layer chromatography that in which the stationary phase is a thin layer of an adsorbent such as silica gel coated on a flat plate. It is otherwise similar to paper chromatography.

af·fin·i·ty chro·ma·tog·ra·phy

chromatography where the absorbent has a unique chemical affinity for a particular component of the passing solution.

Affinity Chromatography

A type of adsorption chromatography used in analytical chemistry in which a biomolecule—e.g., an antigen or antibody—is purified based on a substance's highly specific and reversible adsorption by a complementary binding substance (ligand) that has been previously immobilised on an insoluble support matrix.

chromatography

a technique for analysis of chemical substances. The term chromatography literally means color writing, and denotes a method by which the substance to be analyzed is poured into a vertical glass tube containing an adsorbent, the various components of the substance moving through the adsorbent at different rates, according to their degree of attraction to it, and producing bands of color at different levels of the adsorption column. The term has been extended to include other methods utilizing the same principle, although no colors are produced in the column.

The mobile phase of chromatography refers to the fluid that carries the mixture of substances in the sample through the adsorptive material. The stationary phase (or adsorbent) refers to the solid material that takes up the particles of the substance passing through it. Kaolin, alumina, silica and activated charcoal have been used as adsorbing substances or stationary phases.

Classification of chromatographic techniques tends to be confusing because it may be based on the type of stationary phase, the nature of the adsorptive force, the nature of the mobile phase, or the method by which the mobile phase is introduced.

The technique is a valuable tool for the research biochemist and is readily adaptable to investigations conducted in the clinical laboratory. For example, chromatography is used to detect and identify in body fluids certain sugars and amino acids associated with inborn errors of metabolism.

adsorption chromatography

that in which the stationary phase is an adsorbent.

affinity chromatography

a method of chromatography that utilizes the biologically important binding interactions that occur on protein surfaces. For example, an enzyme substrate is covalently coupled to an inert matrix such as a polysaccharide bead. The enzyme can be bound to the bead and thereby separated when present in very low concentration in a very complex mixture of other macromolecules.

column chromatography

the technique in which the various solutes of a solution are allowed to travel down a column, the individual components being adsorbed by the stationary phase. The most strongly adsorbed component will remain near the top of the column; the other components will pass to positions farther and farther down the column according to their affinity for the adsorbent. If the individual components are naturally colored, they will form a series of colored bands or zones.

Column chromatography has been employed to separate vitamins, steroids, hormones and alkaloids and to determine the amount of these substances in samples of body fluids.

exclusion chromatography

that in which the stationary phase is a gel having a closely controlled pore size. Molecules are separated based on molecular size and shape, smaller molecules being temporarily retained in the pores.

gas chromatography

a type of chromatography in which the mobile phase is an inert gas. Volatile components of the sample are separated in the column and measured by a detector. The method has been applied in the clinical laboratory to separate and quantify steroids, barbiturates and lipids.

gas-liquid chromatography

gas chromatography in which the substances to be separated are moved by an inert gas along a tube filled with a finely divided inert solid coated with a nonvolatile substance; each component migrates at a rate determined by its solubility in the stationary phase and its vapor pressure.

gel-filtration chromatography, gel-permeation chromatography

exclusion chromatography.

high performance liquid chromatography (HPLC)

a miniaturized method in which the solution to be analyzed is passed, under high pressure, through a long, thin column packed with tiny beads such that analyses are completed in minutes rather than hours and with improved resolution.

ion-exchange chromatography

that utilizing resins to which are coupled either cations or anions that will exchange with other cations or anions in the material passed through their meshwork.

molecular sieve chromatography

exclusion chromatography.

paper chromatography

a form of chromatography in which a sheet of special paper is substituted for the adsorption column. After separation of the components as a consequence of their differential migratory velocities, they are stained to make the chromatogram visible. In the clinical laboratory paper chromatography is employed to detect and identify sugars and amino acids.

partition chromatography

a form of separation of solutes utilizing the partition of the solutes between two liquid phases, namely the original solvent and the film of solvent on the adsorption column.

thin-layer chromatography

that in which the stationary phase is a thin layer of an adsorbent such as silica gel coated on a flat plate. It is otherwise similar to paper chromatography.

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